Background
Caffeine, a nonselective adenosine A1 and A2A receptor antagonist, is the most widely used psychoactive substance in the world. In spite of debate about the abuse potential of caffeine, a literature review of human caffeine withdrawal has provided sufficient evidence to warrant the inclusion of caffeine withdrawal as a chemical dependent disorder
Evidence indicates that mesolimbic dopaminergic pathway mediates the reinforcement and behavioral sensitization of caffeine. Many studies also suggest that caffeine interacts with the nigrostriatal dopaminergic pathway to modulate its motor-stimulating effect. The anatomical and functional interactions between the adenosine and dopamine receptors in the striatum have been recently reviewed
Interestingly, two large prospective epidemiological studies have linked coffee drinking to a reduced risk of developing Parkinson's disease (PD)
Drug reward and voluntary motor movement are the two main functions of the dopamine system. Thus, dopamine modulation is central to the disorders of drug addiction and PD. The striatum is the main receiving area of the basal ganglia, and about 95% of the efferent striatal neurons consist of GABAergic medium spiny neurons. These neurons receive a modulatory input from midbrain dopaminergic neurons. The ventral striatum, comprised of the nucleus accumbens, receives its dopaminergic input from the ventral tegmental area and this projection constitutes the mesolimbic pathway, which is involved in drug reinforcement, addiction, and behavioral sensitization
Since caffeine and selective A2A antagonists induce the reinforcement and sensitization behaviors, and exhibit the therapeutic effects in animal models of PD, which are mediated by mesolimic and nigrostrial dopaminergic pathways projected to the striatum, it is reasonable to hypothesize that caffeine and selective A2A antagonists can modulate the neuroadaptation of dopaminergic neurons in the striatum. Indeed, the expression of adenosine A2A receptors in the brain is mostly limited to the striatum
Sensitization of locomotor activity and conditioned place preference are the most commonly studied paradigms, which reflect the incentive motivational properties of drugs believed to contribute to the intensification of drug craving and compulsive drug-seeking behavior
Materials and methods
Animals
Male C57BL/6 mice, purchased from the National Laboratory Animal Breeding and Research Center (Taipei, Taiwan), were established at the Laboratory Animal Center, Tzu Chi University. Mice weighing 25-35g were used in the present study. All experimental procedures were carried out in accordance with the guidelines of the Institutional Animal Care and Use Committee of Tzu Chi University. Every effort was made to minimize the suffering and the number of animals used.
Drugs
Caffeine, DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and SCH-58261 (5-amino-7-(β-phenylethyl)-2-(8-furyl) pyrazolol [4,3-e] - 1,2,4 - triazolol [1,5-c] pyrimidine) were purchased from Sigma-RBI (Taipei, Taiwan). Caffeine was dissolved in saline whereas SCH 58261 and DPCPX were dissolved in dimethyl sulfoxide (DMSO). All drugs were administered i.p. with the dosages specified in each experiment.
Evaluation of locomotor activity
Locomotor activity was monitored as described previously (Hsu et al., 2009). Briefly, a 2-hr habituation period was routinely used prior to the administration of test drugs. Images of the locomotor activity (distance traveled) were captured by a video camera and the recorded images were transferred to the interface of a computer for processing. The track data stored in a special format were retrieved and analyzed by TrackMot software (Diagnostic & Research Instruments Co., Taoyuan, Taiwan). The activity was summated consecutively for three 10-min intervals following the drug administration. In addition, the total distance traveled for the initial 30 min was also summated for analysis. All animals were used only once.
Locomotor sensitizing effects following chronic caffeine and SCH 58261 administrations
According to our previous study (Hsu et al., 2009), dosages of caffeine (10 mg/kg) and SCH58261 (2 mg/kg), which induced conditioned place preference, were used in the chronic sensitization experiments. Mice were administered i.p. caffeine or SCH58261 for 5 consecutive days, and after one-day washout, the locomotor activity was monitored for 30 min by administering an acute dose of caffeine (10 mg/kg) or SCH58216 (2 mg/kg) on day 7. Mice were kept on the same dosages of caffeine or SCH58261 for another 4 consecutive days followed by 3-day washout. The locomotor activity on day 15 elicited by an acute dosage of caffeine (10 mg/kg) or SCH58261 (2 mg/kg) was recorded for 30 min. Animals in the control groups received either saline or DMSO. Acute motor stimulating effects of caffeine (10 mg/kg) or SCH58261 (2 mg/kg) on day 1, 7 and 15 were also recorded in the caffeine-treated groups or SCH58261-treated groups for comparison.
Cross-sensitization effect of caffeine on chronic SCH58261- and DPCPX-treated mice
Mice were administered SCH 58261 (2 mg/kg, i.p.), DPCPX (3 mg/kg, i.p.) or DMSO daily for 14 days. Three days after the last scheduled administration, the locomotor activity of an acute dosage of caffeine (10 mg/kg, i.p.) was recorded for 30 min following 2 hrs habituation. The locomotor activities produced by an acute dosage of caffeine (10 mg/kg, i.p.) between SCH 58261-treated and DMSO-treated groups and between DPCPX-treated and DMSO-treated groups after washout period were compared to assess the locomotor cross-sensitization effect.
Measurement of dopamine concentration in the striatum of sensitized-mice
Following 3-day washout, mice chronically treated with caffeine, SCH58261, or vehicles were sacrificed by decapitation 30 min after an acute corresponding dosage of caffeine (10 mg/kg) or SCH58261 (2 mg/kg). The brains were removed and placed on an ice-cold surface, and the striata were dissected out immediately under a microscope, weighed, and homogenized in the buffer (ice-cold 0.1 M HCl, 0.1 mM sodium metabisulfate). After centrifugation at 12,000 rpm for 10 min, 100 μl of supernatant was removed and further separated using 0.2 μm pore size filter (Millipore, MA, USA) and centrifuged again at 12,000 rpm for 10 min. Dopamine (DA) and its metabolites DOPAC and HVA in the filtrate were quantitated by reverse-phase high performance liquid chromatography (HPLC) with electrochemical detection
Western blotting
Following 3-day washout, mice chronically treated with caffeine, SCH58261, or vehicles were sacrificed by decapitation 30 min after an acute corresponding dosage of caffeine (10 mg/kg) or SCH58261 (2 mg/kg). The brains were then removed and the striata were dissected under a microscope on an ice-cold surface and homogenized in the lysis buffer (0.5 mM dithiothreitol, 0.2 mM EDTA, 20 mM HEPES, 2.5 mM MgCl2, 75 mM NaCl, 0.1 mM Na3VO4, 50 mM NaF, 0.1% Triton X-100, and a cocktail tablet containing protease inhibitors (Roche, Mannheim, Germany)). After centrifugation at 12,000 rpm for 30 min, the supernatant was removed and stored at -80°C until assayed. Protein concentrations were determined using the Bio-Rad protein assay kit. Eighty micrograms of protein from each sample were subjected to 10% SDS-polyacrylamide gel electrophoresis followed by electrophoretic transfer to polyvinylidene difluoride membranes. The membranes were immunoblotted using primary antibodies for phospho-Ser31-TH (1:500) (Abcam; Cambridge, UK), total TH (1:2000) (Abcam) or actin (1:10000) (BD Biosciences; US) and followed with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz; Santa Cruz, CA). Finally, the protein bands were visualized on the X-ray film using the chemiluminescence detection system (ECL, Amersham, Berkshire, England). The intensity of the band was quantified with a densitometric analysis (GS-800 Calibrated Densitometer, Bio-Rad), and calculated as the optical density × area of band.
Statistical analysis
The locomotor activity was calculated for every 10-min recording. In addition, total drug-induced locomotor activities for the entire 30 min on day 1, day 7 and day 15 following drug administrations were summated. Data were expressed as mean ± standard error of the mean (SEM). Data were analyzed for statistical significance using the computer program Prism for two-way ANOVA followed by Bonferroni post-test. In addition, mean ± SEM of total locomotor activity (30 min) was calculated and analyzed by Student's t-test. The concentration of dopamine and its metabolites were normalized by internal standard, and the phosphorylation of TH and total TH were normalized by actin in the striatal homogenates. Data were expressed as mean ± SEM and analyzed by Student's t-test. In all cases, p < 0.05 was considered statistically significant.
Results
Locomotor sensitization after chronic caffeine or SCH58261 treatment
The sub-maximal effective dosage of caffeine (10 mg/kg), which induced conditioned place preference (Hsu et al., 2009), was selected to investigate its locomotor sensitization. Mice were given a total of 10 injections with washout on day 6 and day12 to day 14. Three days after the last treatment with caffeine or saline, acute administration of caffeine (10 mg/kg i.p.) caused a greater locomotor response from caffeine- vs vehicle-pretreated mice (Fig.
Locomotor sensitization by repeated caffeine and SCH58261 administration in habituated C57BL/6 mice
Locomotor sensitization by repeated caffeine and SCH58261 administration in habituated C57BL/6 mice. Caffeine (10 mg/kg/day, i.p.), SCH58261 (2 mg/kg/day, i.p.) or vehicles were administered continuously for 10 days except with one washout on day 6. Three days after the last injection of Caffeine, SCH58261 or vehicles, mice were challenged with caffeine (10 mg/kg, i.p.) or SCH58261 (2 mg/kg/day, i.p.) respectively. The horizontal locomotor activity was measured for 30 min. Data represent means ± SEM (n = 4-6). a, d The time course of locomotor activity measured over 10-min intervals. a caffeine-treated group compared to saline-treated group(P < 0.001) and d SCH58261-treated group compared to DMSO-treated group (P = 0.003, by two-way ANOVA); b, e Total locomotor activity counts during the 30-min period following acute administration of caffeine or SCH58261. b caffeine-treated group compared to saline-treated group (P < 0.01)and e SCH58261-treated group compared to DMSO-treated group (P < 0.05); c, f Total locomotor activity counts during the 30-min period following acute administration of caffeine in caffeine- treated group and acute administration of SCH58261 in SCH58261-treated group on day1, day7 and day15. c locomotor activity of caffeine-treated group on day7 and on day15 compared to locomotor activity of caffeine-treated group on day1, and f locomotor activity of SCH58261-treated group on day7 and on day15 compared to locomotor activity of SCH58261-treated group on day1 respectively (*P < 0.05 and **P < 0.01 by Student's test).
The sub-maximal effective dosage of SCH 58261 (2 mg/kg), which induced conditioned place preference (Hsu et al., 2009), was used to study the locomotor sensitization. The protocol for chronic SCH58261 treatment was analogous to that of caffeine. Three days after the last injection of DMSO or SCH 58261, acute administration of SCH 58261 (2 mg/kg i.p.) resulted in a greater response in the locomotor activity from SCH 58261- as compared with vehicle-pretreated mice. (Fig.
Cross-sensitization between Caffeine and SCH58261 but not between caffeine and DPCPX
After the last injection of SCH 58261 and followed by a 3-day washout period, acute challenge with caffeine (10 mg/kg i.p.) caused a greater response in the locomotor activity from SCH 58261- vs vehicle-pretreated mice. (Fig.
Cross-sensitization by repeated administration of SCH58261 or DPCPX in habituated C57BL/6 mice
Cross-sensitization by repeated administration of SCH58261 or DPCPX in habituated C57BL/6 mice. SCH58261 (2 mg/kg/day, i.p.), DPCPX (3 mg/kg/day, i.p.) or DMSO was administered for 14 days. Three days after the last injection of SCH58261, DPCPX or DMSO, mice were challenged with caffeine (10 mg/kg, i.p.) The horizontal locomotor activity was measured for 30 min. a The time course of locomotor activity measured over 10-min intervals. Data represent means ± SEM (n = 3). P < 0.001 versus DMSO-treated group (by two-way ANOVA). b Total locomotor activity counts during the 30-min period following acute administration of caffeine. Data represent means ± SEM. P < 0.01 versus DMSO-treated group (by Student's test). c The time course of locomotor activity measured over 10-min intervals. Data represent means ± SEM (n = 4). Data showed no significant difference between DPCPX-treated and DMSO-treated group.
Chronic caffeine and SCH58261 administrations were associated with significant changes in monoamine systems in the striatum
The effect of chronic caffeine and SCH58261 administrations on striatal amines were shown in Fig.
Effect of chronic caffeine and SCH58261 administration on the striatal amines (n = 3-4)
Effect of chronic caffeine and SCH58261 administration on the striatal amines (n = 3-4). a DA leves in the striatum after chronic caffeine treatment was increased. Data represent means ± SEM. P < 0.01 versus saline-treated group (by Student's test). b, c Increase in DOPAC and HVA levels were also noted but were not statistically significant. d DA level in the striatum after chronic SCH58261 treatment was increased. Data represent means ± SEM. P < 0.01 versus DMSO-treated group (by Student's test). e, f Increase in DOPAC and HVA levels were also noted. P < 0.001 and P < 0.05 versus DMSO-treated group respectively (by Student's test).
Chronic treatment with caffeine and SCH58261 increased TH phosphorylation at Ser31 in the striatum
Mice were treated with caffeine (10 mg/kg, i.p.) or SCH58261 (2 mg/kg, i.p.) for 10 days as described for the locomotor sensitization experiments. Following 3-day washout period, mice were sacrificed 30 min after acute challenge with caffeine (10 mg/kg) or SCH58261 (2 mg/kg, i.p.). Striatal membrane was prepared for the Western blotting of total TH and phosphor-Ser31-TH expression. As shown in Fig
Representative Western immunoblots of phsopho-S31-TH (a, c) and total TH (b, d)in the striatum of the chronic saline- or caffeine-treated groups and chronic DMSO- or SCH58261-treated groups 30 min after a challenge with caffeine (10 mg/kg) or SCH58261 (2 mg/kg)
Representative Western immunoblots of phsopho-S31-TH (a, c) and total TH (b, d)in the striatum of the chronic saline- or caffeine-treated groups and chronic DMSO- or SCH58261-treated groups 30 min after a challenge with caffeine (10 mg/kg) or SCH58261 (2 mg/kg). The experiment was repeated at least three times. The
Discussion
Our previous and other studies have demonstrated that moderate dosages of caffeine (15 and 20 mg/kg) induce locomotor sensitization. However, conditioned place preference was not reported with these dosages of caffeine. Instead, low dosage of caffeine (10 mg/kg), which is more in line with the amount normally ingested in beverages and food, can induce conditioned place preference but the locomotor sensitization has not been reported
Locomotor sensitization, proposed to reflect the increase of the wanting for drug reward, would result from an increase of the responsiveness of dopaminergic neurons to stimuli
Our results also showed that chronic treatments with caffeine and SCH58261 increased the dopamine concentration and TH phosphorylation at Ser31 in the striatum in caffeine- and SCH58261-sensitized mice. Indeed, it has also been reported that 10 mg/kg of caffeine can reverse the catalepsy and decrease the activity produced by DA antagonists in rats
Most studies about caffeine and A2A antagonists focus on the neuroprotection against dopaminergic neurodegeneration in animal models of PD
We also demonstrated that chronic treatment of caffeine and a selective A2A antagonist enhance the phosphorylation level of tyrosine hydroxylase at Ser31. Phosphorylation of TH is likely to be of physiological importance in maintaining catecholamine stores because TH is the rate-limiting enzyme in catecholamine biosynthesis and its activity is increased by phosphorylation
In vitro, caffeine at mM concentrations can activate tyrosine hydroxylase in bovine chromaffin cells
In conclusion, our study demonstrates that low dosages of caffeine and a selective adenosine A2A antagonist SCH58261 induce sensitization and cross-sensitization of locomotor activity, which are associated with elevated dopamine concentration and phosphorylation of TH at Ser31 in the striatum. Blockade of adenosine A2A receptor may play an important role in the striatal neuroadaptations observed in the caffeine- and SCH58261-sensitized mice.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CWH and CSW performed animal and pharmacological experiments and the acquisition of data. CWH and THC participated in the experimental conception and design, and were also involved in the interpretation of data, drafting and revising the manuscript.